CD4 T cells play a central role in orchestrating adaptive immune responses. After being activated through their T cell receptor (TCR) in a particular cytokine milieu, naive CD4 T cells differentiate into distinct T helper (Th) lineages, including Th1, Th2 and Th17 cells that produce interferon (IFN)-gamma;, interleukin (IL)-4 and IL-17, respectively, as their signature effector cytokines. These cells are indispensable for different types of immunity to various microorganisms: Th1 cells are important for protective immune responses to intracellular viral and bacterial infection; Th2 cells for expelling extracellular parasites such as helminths; and Th17 cells for controlling extracellular bacteria and fungi. Additional CD4 T cells include follicular T cells (Tfh) and regulatory T cells (Treg). Tfh cells are critical for promoting antibody responses, whereas Treg cells, which consist of natural-occurring regulatory T cells (nTregs) and inducible regulatory T cells (iTregs), are responsible for maintaining immune tolerance and lymphocyte homeostasis. Inappropriate Th responses to pathogens may lead to chronic infection and/or tissue damage to the host. Similarly, unnecessary activation of Th1, Th17 or Th2 cells by harmless environmental- or self-antigens can cause organ-specific autoimmune diseases or allergic inflammatory diseases. The activation, differentiation and expansion of Th cells are tightly regulated by specific transcription factors that are induced and/or activated by a combination of cytokines. During Th1 cell differentiation, IFNgamma and IL-12 are responsible for inducing T-bet gene expression and Stat4 phosphorylation, respectively; both T-bet and Stat4 are critical for Th1 cells to produce IFNgamma. Similarly, GATA-3 expression and Stat5 activation induced by IL-4 and IL-2, respectively, are the two key elements for Th2 cell differentiation and functions. RORgammat/Stat3, Foxp3/Stat5, and Bcl-6/Stat3 are the crucial counterparts during differentiation of Th17, Treg and Tfh cells, respectively. However, these so-called master regulators, although necessary, may not be sufficient to induce a specific Th cell phenotype. Many other transcription factors, forming a network with these key factors, also act in the process of T cell differentiation. In addition, for these known transcription factors, how they regulate lineage-specific gene expression and/or epigenetic modifications through their binding to cis-regulatory elements are still elusive. Therefore, identifying the components of transcriptional regulatory network and investigating mechanisms of their actions are essential for understanding the differentiation processes into distinct Th cell lineages. Unit scientists have identified GATA3 targets in different T cells genome-wide. GATA3 is not only important for Th2 cell differentiation but is also critical for CD4 T cell development at multiple stages. However, very few genes have been identified as direct targets of GATA3. These genes include Th2 cytokines (IL-4, IL-5, IL-10 and IL-13), a Th2-specific cytokine receptor (T1/ST2, also known as IL-33R) and a transcription factor critical for CD4 T cell development (Th-POK). To study the gene regulatory network involving GATA3 at a systemic level, in collaboration with Dr. Keji Zhaos lab, we mapped GATA3-binding sites and identified genes regulated by GATA3 in different types of T cells at a genome level via ChIPseq and RNAseq analyses. Through analyzing massive genomic data obtained in this study, we uncovered hundreds of genes that are directly or indirectly regulated by GATA3, including all the known targets. In Th2 cells, GATA3 positively or negatively regulates many transcription factors some of which are known to be important for Th cell differentiation and cytokine production. These transcription factors may form an important transcriptional regulatory network to modulate the expression of Th2-specific genes that are also revealed in this study including cytokines, chemokines, cytokine receptors, chemokine receptors, other cell surface molecules, and signaling molecules. The study also demonstrates that GATA3 binding to DNA and its function in gene regulation are highly dependent on cell context, possibly due to differential expression of GATA3 co-factors in different cells or at different stages. Interestingly, deletion of Gata3 only affects the expression of 10% of the genes bound by GATA3. In addition, several genes that are regulated by GATA3 only in a specific cell type are bound by GATA3 in many other cells. These data indicate that DNA binding and gene regulation are dissociated and that GATA3-mediated gene transcription is more cell type specific than GATA3 binding to DNA. This finding may represent a general principle of gene regulation applicable to many other transcription factors. GATA3 is also expressed in regulatory T cells (Tregs) at intermediate levels, lower than in Th2, but much higher than in Th1, cells. GATA3 ChIPseq study revealed that GATA3 binds to the Foxp3 locus in a Treg-specific manner. Additionally, GATA3 binds to the Tbx21 and the Rorc locus in Tregs. In collaboration with Yasmine Belkaids lab, we studied the function of GATA3 in Treg cells using GATA3 conditional knockout mice. GATA3 expression is upregulated in Tregs by T cell activation and IL-2 stimulation. Deletion of the Gata3 gene in Tregs by either OX40Cre or Foxp3Cre results in reduced Treg cell number as well as reduced Foxp3 expression levels in an inflammatory environment. GATA3-deficient Tregs fail to suppress diseases developed as a result of transferring nave CD4 T cells into Rag2-/- mice. Strikingly, GATA3 and RORgammat expression are mutually exclusive in Treg cells. GATA3-deficient Tregs are more likely to become RORgammat+ and thus IL-17-producing cells during inflammatory responses, whereas enforced GATA3 expression prevents Tregs from becoming IL-17-producing cells. Thus, GATA3 is critical for maintaining Treg cell number and Foxp3 expression and for restraining Treg cell plasticity. We have developed a T-bet-ZsGreen reporter (TBGR) mouse strain, in which GFP faithfully reflects T-bet expression. After cross-breeding TBGR with a number of mouse strains deficient in key Th1 factors, such as Stat4, IFNgammaRI and T-bet, alone or in combination, we studied the regulation and functions of T-bet in vivo in responses to T. gondii infection. We found that signals elicited by IL-12 and IFNgamma are redundant in inducing T-bet. There is a third previously unrecognized signal that induces T-bet through an IL-12- and IFNgamma-independent mechanism in mice infected with Toxoplasma gondii. T-bet is not required for its own expression when induced by IL-12 and IFNgamma. While both T-bet and Stat4 are critical for IFNgamma production, IFNgamma signaling is dispensable. Loss of T-bet results in activation of an endogenous Th2 program in cells expressing T-bet-ZsGreen. We also performed genome-wide analyses to identify T-bet binding sites and its responsive genes. This study demonstrated redundancy and synergy among several Th1-inducing pathways in regulating Th1 responses and highlighted a critical role of T-bet in suppressing an endogenous (default) Th2 program by inhibiting GATA3 expression and function during ongoing Th1 responses. Our study implies that, by contrast to Th1 and Th17 differentiation requiring instructive signals from antigen-presenting cells, Th2 responses can be triggered through a default pathway.